Fibroblast growth factors (FGFs) comprise a family of at least 24 multifunctional polypeptides involved in a variety of biological processes including morphogenesis, angiogenesis, and tissue remodeling as well as in the pathogenesis of numerous diseases (for review see Ornitz, Bioessays 22, 108, 2000). The various members of this family stimulate the proliferation of a wide spectrum of cells, ranging from mesenchymal to epithelial and neuroectodermal origin in vitro and in vivo. Types of cells responding to FGF mitogenic stimuli include fibroblasts, corneal and vascular endothelial cells, granulocytes, adrenal cortical cells, chondrocytes, myoblasts, vascular smooth muscle cells, lens epithelial cells, melanocytes, keratinocytes, oligodendrocytes, astrocytes, osteoblasts, and hematopoietic cells. FGFs are expressed in a strict temporal and spatial pattern during development and have important roles in patterning and limb formation (Ornitz et al., J. Biol. Chem. 271, 15292, 1996).
FGFs are powerful mitogens and are critical in regulation of many biological processes including angiogenesis, vasculogenesis, wound healing, limb formation, tumorigenesis and cell survival. The biological response of cells to FGF is mediated through specific, high affinity (Kd 20-500 pM) cell surface receptors that possess intrinsic tyrosine kinase activity and are phosphorylated upon binding of FGF (Coughlin et al. J Biol. Chem. 263, 988, 1988). Five distinct Fibroblast Growth Factor Receptors (FGFRs) have been identified (Johnson and Williams, Adv. Cancer Res. 60, 1993; Sleeman et al., Gene 271, 171, 2001). The FGFR extracellular domain consists of three immunoglobulin-like (Ig-like) domains (D1, D2 and D3), a heparin binding domain and an acidic box. Alternative splicing of the FGF receptor mRNAs generates different variants of the receptors. In general, the FGF family members bind to all of the known FGFRs, however, some FGFs bind to specific receptors with higher degrees of affinity. The FGFR genes have been cloned and identified in mammals and their homologues described in birds, Xenopus and Drosophila (Givol and Yayon, FASEB J. 6, 3369, 1992).
Another critically functional component in receptor activation is the binding to proteoglycans such as heparan sulfate. FGFs fail to bind and activate FGF receptors in cells deprived of endogenous heparan sulfate. Different models have been suggested in attempts to explain the role of heparan sulfate proteoglycan (HSPG) in FGF signaling, including the formation of a functional tertiary complex between FGF, FGFR and the appropriate HSPG (Yayon et al., Cell 64, 841, 1991).
A number of birth defects are associated with mutations in the genes encoding FGF receptors. For example a mutation in FGFR1 is associated with Pfeiffer syndrome. Certain other mutations in FGFR2 are associated with Crouzon, Pfeiffer, Jackson-Weiss, Apert or Beare-Stevenson syndromes. The clinical manifestation of Apert syndrome (AS) is characterized by both bony and cutaneous fusion of digits of the hands and the feet. Broad thumbs and halluces distinguish Pfeiffer syndrome, while in Crouzon syndrome limbs are normal but a high degree of proptosis is evident. The most prominent malformation syndrome associated with these mutations is craniosynostosis (the premature fusion of the skull bones sutures). Mutations in FGFR3 are responsible for achondroplasia, the most common form of human genetic dwarfism. Thanatophoric dysplasia is a severe and lethal form of FGFR3 mutations, while hypochondroplasia is a milder form of achondroplasia. Examination of the sequence of FGFR3 in achondroplasia patients identified a mutation in the transmembrane domain of the receptor (reviewed in Vajo et al., Endocrine Rev. 21, 23, 2000).
The FGFRs have been implicated in certain malignancies. FGFR3 is the most frequently mutated oncogene in bladder cancer where it is mutated in more than 30% of the cases (Cappellen et al., Nature Genet. 23, 18, 1999). Recently, Dvorakova et al. (Br. J. Haematol. 113, 832, 2001) have shown that the FGFR3IIIc isoform is overexpressed in the white blood cells of chronic myeloid leukemia (CML) patients. Yee et al. (J. Natl. Cancer 92, 1848, 2000) have identified a mutation in FGFR3 linked to cervical carcinoma.
A great deal of work was invested in structure-function studies of FGFs and their receptor binding elements. These have led to the determination of the major and minor receptor binding domains, heparin-binding residues, peptomimetics having structures based on FGFs and FGF peptides that were constructed from phage-display technology.
It has been well characterized that some FGFs, such as FGF-1, stimulate all of the receptor isoforms, however, some FGFs bind specifically to selected receptors with orders of magnitude higher affinities. Specificity may also be achieved by other factors, such as different proteoglycans, expressed in different tissues (Ornitz, Bioessays, 22, 108, 2000). Recently, site-directed mutagenesis and X-ray crystallography were used to investigate the basis of specificity of FGFs to their receptors. These were based mostly on the structures of the extracellular domain of FGFR1 and FGFR2 bound to FGF-1 and FGF-2 (Plotnikov et al., Cell 98, 641, 1999; Plotnikov et al., Cell 101, 413, 2000; Stauber et al., PNAS USA 97, 49, 2000; Pellegrini, et al., Nature, 407, 1029, 2000; Schlessinger et al., Mol Cell, 6, 43, 2000).
Generation of specific ligands would be useful for the purpose of research as well as for the purpose of developing possible medicaments for treatment of diseases and disorders including tumor progression, skin lesions, neurodegenerative diseases, bone fracture healing, achondroplasia, and other skeletal disorders. Additionally, the focus of FGFR3 as the receptor involved in achondroplasia, as well as in cancer including but not limited to transitional cell carcinoma (TCC) of the bladder, multiple myeloma, chronic myeloid leukemia (CML) and cervical carcinoma has raised the unmet need for ligands specific for this receptor, which do not substantially bind to the other four FGFRs. In light of the large number of FGFs and receptor variants, a major question regarding FGF function is their receptor specificity. In fact, all FGFRs tested so far bind FGF-1 and FGF-4 with moderate to high affinity, demonstrating an apparent redundancy in the FGF system. In contrast to FGFR1 and FGFR2, the third receptor subtype, FGFR3 was found to bind with high affinities to FGF-8, FGF-17 and FGF-18 and with improved selectivity to FGF-9. Producing FGF ligands with enhanced receptor selectivity, higher stimulative activity in vivo, and ease of expression mode, is highly needed for treatment of various pathological conditions.
Attempts have been made to alter FGF receptor specificity by deletions or truncations of its ligands, by means of mutations introduced at certain locations within the gene encoding for the proteins. Mutations affecting the binding affinity as well as binding to heparin have been demonstrated by several investigators. For example Seno et al. (Eur. J. Biochem. 188, 239, 1990) studied the effect of the carboxy and amino termini of basic FGF on the affinity for heparin. Truncation of more than 6 amino acids from the C-terminus of bFGF decreased the affinity for heparin, though removal of up to 46 amino acids showed a significant stimulation of the proliferative effect. Removal of 40 amino acids from the N-terminus exhibited comparable affinity to heparin as that of intact bFGF, and induced stimulation of DNA synthesis.
Additional truncated versions of molecules of the FGF family have been reported by Kuroda et al., (Bone, 25, 431, 1999). Kuroda et al., produced amino terminus truncated human FGF-4 of various sizes, and tested the effects on growth stimulation and increase in bone density. The full-length polypeptide, and a shortened version containing 134 amino acid residues demonstrated comparable cellular proliferation and effect on increase of bone density. The shortest form of FGF-4 tested, containing only 111 amino acid residues exhibited limited growth stimulatory activity.
A spontaneous truncation of 34 amino acid residues, including the methionine residue encoded by the initiation codon, was discovered in the N-terminus of FGF-16 expressed in E. coli. The variant retained biological activity as measured as induction of cell proliferation in vitro as well as in vivo (Danilenko et al., Arch. Biochem. Biophys. 1, 361, 1999). In addition, FGF-16 having from one to thirty-four amino acids deleted from the N-terminus or from one to eighteen amino acids deleted from the C-terminus, was shown to retain biological activity (U.S. Pat. No. 5,998,170).
The human FGF-9 gene was found to code for a 208 amino acid protein, which shares approximately 30% homology with other FGFs and presents a unique spectrum of biological activity as it stimulates the proliferation of glial cells, PC-12 cells and BALB/C 3T3 fibroblasts, but not endothelial cells (U.S. Pat. Nos. 5,622,928, and 5,512,460). A 152 amino acid fragment of the FGF-9 comprising a truncation of 53 amino acids from the N-terminus and 13 amino acids from the C-terminus is further disclosed in U.S. Pat. No. 5,512,460. Deletion of 54 amino acids from the N-terminus of the protein yielded a 154 amino acid protein retaining its biological activity (U.S. Pat. No. 5,571,895).
Basic FGF (FGF-2) has been modified to alter biological properties and binding specificity. U.S. Pat. No. 5,491,220 discloses structural analogues of FGF-2 that comprise substitution of the β9-β10 loop with that of another FGF or IL-1β to alter biological properties and binding specificity. Human FGF-2 (basic FGF) has been designed with substitutions at either one or more of the following amino acids glutamate 89, aspartate 101 and/or leucine 137, which impart beneficial therapeutic properties (U.S. Pat. No. 6,274,712). U.S. Pat. No. 6,294,359 discloses analogs of FGF-2 that comprise amino acid substitutions at heparin and receptor binding domains. The patent presents analogs that are either agonist or antagonist with respect to wild type FGF in a cell proliferation assay but does not teach receptor specificity changes.
Mutant forms of FGF-10 (also known as KGF-2) including amino and carboxy terminal truncations and amino acid substitutions have been disclosed in U.S. Pat. No. 6,077,692. The patent discloses variants that exhibit enhanced activity, higher yields or increased stability but neither teaches nor suggests a change in receptor specificity.
WO 01/39788 discloses targeting cells expressing FGFR2 or FGFR3 by using compositions comprising FGF-18.
The extensive efforts made to produce truncation, deletion and point mutation variants in FGF have resulted in changes in affinity to the receptors but not in significant alterations in receptor specificity. Thus, there is an unmet need for highly active and selective ligands for the various types of FGF receptors that would be useful in stimulation or inhibition of these receptors thereby addressing the clinical manifestations associated with the above-mentioned mutations, and modulating various biological functions.
It is explicitly to be understood that known active fragments of FGFs are excluded from the present invention.